17 – Compounds that Regulate SynGAP1 Protein Expression

Event Time

December 4, 2020 at 5:00 pm

Description

Here are our introductory comments:

Dr. Rumbaugh has intensively studied Syngap1 over his long career.  From a cellular perspective Syngap1 has two major biological functions: one in synapse function, and another in cellular morphogenesis (position and shape) .  In Syngap1 Syndrome, both these functions are altered, changing how postsynaptic dendrites mature and also how neural circuits work.  As a result patients have many symptoms, including changes in sensory processing and behaviors.

Dr. Rumbaugh developed a high-throughput screen to find drugs that upregulate Syngap1 expression.  We are lucky that he has a position at Scripps in Jupiter, Florida, home of one of the biggest drug libraries in existence.  While no drugs which directly upregulate Syngap1 expression have been found (to date), several drugs down-regulate expression.  By following these leads, biological pathways can be found that might be modified by a drug, in order to increase the steady-state levels of Syngap1 protein.  SRF is supporting some of Dr. Rumbaugh’s work with a full time three year postdoctoral fellow. Some of their work includes the development and sharing of a test to measure the level of Syngap1 protein.  This assay will be useful to researchers working to increase the expression levels of Syngap1, which is the goal of all the therapeutics currently in development by various research and corporate groups.

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0:03we’re going to move on to our next speaker and thank you to the speakers we are right on time so this is great

0:09so our next speaker is dr gavin rumball he is a professor at the scripps research institute in florida and he his overarching goal

0:18of his lab is to apply knowledge from biological studies of genetic risk factors such as syngap to accelerate development

0:24of therapeutic agents to treat impaired brain excitability and behavioral dysfunction

0:30and disorders associated with autism spectrum intellectual disability epilepsy we’re excited to hear about his

0:36work today thanks gavin

0:44hi can you can you guys hear me and see my screen okay everything looks great oh thanks a lot

0:49thanks for the introduction heather thanks mike and hans for for imitation uh i heard a lot of great things about

0:56the first round table last year and so it’s pleasure uh to be invited and participate uh with

1:02my uh fellow speakers who i you know i know all very well so uh today i’m gonna talk today about

1:10uh our discovery platform for compounds that that regulate synthetic protein expression and i think you got a

1:15hint from this from uh from liz’s talk that it’s the goal of many of ours because of the nature of the genetic disorders

1:21think up one genetic disorder that we think raising uh finding ways to regulate syngat protein expression in brain cells

1:28will be an important way to treat cingapians so today i’m going to talk about our a little bit more detail about this platform that we’ve discovered for

1:35unbiased discovery of compounds that regulate synga protein and neurons

1:40but before i uh there we go sorry about that but before i do that i just want to spend a few minutes on

1:46kind of the way that we conceptualizing kepler disorders you know we’re i’m a basic scientist i’m a neurobiologist neurophysiologist

1:54and my general training and interest is in understanding how genes regulate functional neural circuitry to uh to

2:00carry out important neural domains that we all

2:05cherish such as in memory and planning and language and motor movements and sensory

2:11perception and how these processes may go awry to cause

2:17problems that we see see in the kids including seizure and so you know generally what my lab is

2:23interested in understanding is how does thing gap one gene carry out critical neurobiological functions to

2:28lead to a normally functioning brain and so the the converse of that is how do you when you lose one copy of

2:33syngap1 you know what is the what are the pathological consequences to the brain or what are the functional

2:38consequences to the brain that leads to altered memory planning language uh uh and so on so that’s generally how

2:44we conceptualize uh on the disorder and so you work from many many labs

2:52for you know more than 20 years now has really identified you mary talked about uh three functions

2:57for singap in the synapse from a bio from a biochemical perspective

3:04we see two general functions of syngap1 from us from a cellular perspective and

3:09how these functions of syngap1 may help to organize uh functional circuitry that carry out

3:14important neural domains so as you heard from mary it’s the syngap is an important uh

3:20constituent of uh of the postsynaptic density principally in dendritic spines and in excitatory neurons in the forebrain

3:28and so uh there it regulates a multitude of functions to regulate synaptic strength and we think to normalize both

3:34development of neural circuits uh neural circuit excitability but also neural neural circuit plasticity

3:41and so another function of syngap that we’ve principally uh articulated uh to to the field over

3:48and now in five five different papers is when we look in various areas of the brain we we always see

3:56uh changes in dendritic architecture and so that’s important and we don’t think that importantly we don’t think that these changes in dendritic architecture

4:02are related to alterations in syngap function to synaptic function and the reason is depending on where we look

4:08we may see for instance in in upper laminous uh cortical sensory neurons we see that uh in syngap one uh

4:15heterozygous knockout mice which model our model for the genetic disorder we see under uh uh or what are called

4:22arrested so neurons that underwent arrested development so these are much smaller neurons and they never make it to the right size so they

4:29have fewer connectivity onto them but just a few hundred microns away and deeper uh lamina

4:35of the same part of the cortex you see actually in developing animals you see much more mature much larger dendritic

4:42arbors in syngap1 mutant mice and so you know the point is is that there’s a

4:48function of singap and we don’t understand the molecular functions that lead to the changes in dendritic architecture but the point is that these dendritic

4:55these these changes in dendritic architecture affect the way that functional circuits develop uh uh and these and you can imagine that

5:02alterations in synaptic function and plasticity are superimposed onto a background of altered connectivity of neural circuits

5:09and we think that it’s the combination of these two substrates in the developing brain that leads ultimately to the to the

5:15changes in behavior and uh and adaptive functioning that that defines this uh singapore disorder

5:21and i would mention that we’ve also seen alterations pretty pretty significant alterations in dendritic

5:26uh uh arborization and architecture uh in um uh in human neurons derived from ipscs

5:32that we published just a few months ago okay so the way that we generally conceptualize syn gap on disorders

5:38it’s a developmental encephalopathy where there’s altered uh brain function and principally altered brain function is going to

5:44affect different domains that that are responsible for everything that we do which would include cognition

5:50valence uh whether you know something is good for us or bad for us of course social processes and arousal

5:56and regulatory uh functions that are important for learning and so the way the way that

6:02most genetic neurodevelopmental disorders are conceptualized is that what happens when you lose or when you change the function of these

6:08genes that you alter this functional neural circuitry or functional networks and these changes in functional networks are what underlie

6:15the symptoms that we see and the way that i conceptualize singap1 is that epilepsy is an outcome of this

6:21altered functional neural circuitry so just as altered cognitive function is an outcome

6:26of this i see the epilepsy um also is uh uh as an outcome of this

6:31generalized disruption to functional architecture and so we think principally based on prior studies that

6:36syngap1 is is particularly important in projection neurons from the cortex so so glutamatergic neurons the spiny

6:42neurons expeditory neurons and so we’re trying to better understand how to conceptualize this and just as

6:48just a flavor of what the lab is working on now from the biological perspective is that we’re viewing uh we’re trying to

6:54understand how syngap1 affects functional circuits that are important uh for for how we understand the world

7:00and how we make decisions and we want to do that because after talking to many parents

7:06we realize that there are some altered behaviors and also pretty significant alterations in

7:12sensory functions if you think about sensory function pretty much everything that we do is guided by how we uh how our brains map

7:18the external world and so if we’re if the brains of singapians are unable to process the sensory function you can

7:24imagine that they would have problems uh learning or defending themselves or uh uh

7:29you know basically keeping themselves safe so the way that we’re doing this is to is to is to take a sing up on animals

7:35and uh uh record from various areas of the brain as they do uh what are called ethological tasks or tests

7:41uh that they would normally do in the wild to help survival and so i think some of you have seen some of this recently in other meetings

7:48and so hopefully when i talk to you again in a year or two we’ll be able to show you a more

7:53holistic view of how we think uh singing gap one is affecting distributed brain function but today i’m

7:59going to talk to you principally about this platform uh about how we’re looking for small molecules uh or or drug candidates

8:08that can be used to raise cingup protein expression and why do we want to do this is one of the attractive features that

8:14singap1 from from a therapeutic perspective is that

8:19nearly all of the patients have very clear loss of function barriers meaning that they have genetic haploid

8:24sufficiency where essentially one copy of syngap doesn’t work as well as it should and what you end up with is uh less uh

8:31protein uh in neurons uh uh uh um in the developing basically

8:37throughout life and so having that reduced protein is what ultimately is the root cause of syngap1 disorders

8:43uh in in uh even for patients that don’t have clear loss of function variants those that have very clear clinical

8:49phenotypes some preliminary studies we’ve done in the lab with their stem cells suggests that they also uh indeed express less functional syn

8:55gap so we really do view this as it is as a disease of too little synga protein expression so

9:01therefore um it would be very very important to find therapeutic approaches be targeted

9:06trying to raise uh uh syngat protein levels and that’s one of the reasons why i’m so excited for

9:12people uh new investigators that come into the field like liz heller who has really uh you know whole career is

9:18trying to understand how you regulate uh gene and protein expression so we need expertise like liz to be able to help

9:23solve this uh uh you know this problem of how to raise thing that protein expression so as liz mentioned we know very little

9:30in fact we know almost nothing about how syngap is regulated and so uh you know basically we decided

9:36uh to take a step back and say okay what what might be the ways that we might be able to do this so

9:41so we just conceptualized it as very simply is that this is a disease of low protein expression and that if you have uh this low protein

9:49expression leads to disease phenotypes uh or you know altered uh you know clinical manifestations and so if we

9:55restore protein levels we think that we would be able to improve outcomes and of course we published many papers in animal models doing

10:02genetic gene therapy and showing that we can recover uh important uh um functions of singap

10:08so currently there are various ways that this can be done and and so what we’re working on is what we call plan b

10:14because it’s an alternative uh in a parallel track to to plan a which is being undertaken by

10:19various academic labs around the world as well as several biopharma biopharma companies which is to use asos

10:25or antisense oligonucleotides uh to trick syngap into expressing uh more of its protein it can do this

10:31through skipping poison exons or targeting a natural antisense transcript that might suppress expression of seeing that

10:37and these are very exciting technologies and and i hope that they work but they’re not no

10:43there’s no guarantee that they’re going to work well and even if they work they might not work for all patients and so it’s important to have other ways

10:49to do this and so what we plan to do is to just work on understanding

10:54um you know sort of in the same sense as what liz is trying to do except we’re going to take an unbiased

10:59approach to find perhaps any pathway any druggable pathway that can ultimately lead to increased

11:05sensing gap steady state protein and so there’s many different potential druggable pathways that could

11:11lead ultimately to more syngat protein steady state protein in the cell so you can for instance you can regulate

11:17the amount of mrna that’s made you can alter how that mrna is regulated so more gets

11:22loaded into the uh into the ribosome to make more protein and importantly they’re and they’re all

11:29types of regulatory pathways within the level of initiating translation of mrna and of course we

11:35know in neurons that neural uh that protein degradation is incredibly important for regulating protein levels so there’s uh there are

11:41several documented potential druggable pathways at the level of protein degradation so if you could increase or boost these pathways you

11:48might get more syngap protein or uh for example if you could find a pathway to degradate

11:54the degrade syngap if you inhibited that pathway you might end up with more synthetic protein but since we don’t know anything we

12:00decided to take an unbiased approach and it says if we just created the right kind of assay or the right type of system that

12:06tells us how much syngap is in neurons if we put enough biologically active small molecules

12:12on you know on neurons we might be able to find that one important compound that indeed could raise in that

12:18expression so the ingredients for a successful singup boosting compound program are basically their three

12:26distinct elements one is you need a great assay so you need a great system uh that that is disease modeling and can

12:33tell you how much syngap uh is is inside of brain cells you need a specialized facility with robotics

12:40specializes in what’s called high throughput screening where you can take this great assay and then and then uh use individual

12:47small small molecule compounds tens of thousands of them against this assay to find those ones that do what you want

12:53which is in this case to raise syngap protein and then importantly you need an ecosystem or or a research environment

12:59where you can take a compound in your assay that may raise ingot protein then you need to validate it

13:05biologically validate it to show that it actually raises it in a in a context that’s meaningful and that

13:10can actually change and improve disease related phenotypes in various models whether they be

13:16rodent or or human okay so this system we’ve been working on now for

13:21almost nine years and the reason why is because we had to we had to reverse engineer in some ways higher

13:27throughput screening because we have to do this in neurons and why do we have to do high-throughput screening in neurons is because syngap

13:32while the message may be present in many tissues and i know that i get a lot of questions about this there’s very few document documented

13:39examples of protein existing outside the brain and even within uh within

13:44outside of neurons within the brain so even uh astrocytes with the brain we see very little syngap expression tons of

13:50message very little synthetic protein expression so so in our experience if you wanted to understand

13:56the regulation uh of real syn gap then you would need to measure expression from

14:02uh from neurons and in particular you’d want to do this in neurons that only have one good functioning copy because

14:07it’s very likely that when you lose one copy of singap1 there’s the cell undergoes a state

14:13change where it’s now coping and compensating for having only one functional singup copy and it’s entirely reasonable that the

14:19the druggable pathways that exist in a cell with only one good copy of syngap may be different from the juggable pathways in a normal uh cell that

14:27expresses a hundred percent of synthetic so you would need to do this in neurons that lack one good copy and you would need to be able to track

14:33uh uh track that that the syngat protein in real time that’s made from that one remaining good allele so what we did was

14:39we came up with a system uh what’s called a dual luciferase system where we make a mouse that has uh that expresses a uh one type

14:47of luciferase this just tells you how many neurons uh are in your assay plate and then we

14:52paired that with a with a a specific knock-in mouse when we knocked a luciferase reporter tag with only

14:5811 amino acid tag uh into an internal exon within the syngap1 mouse locus and what you end up

15:05with is a is a tiny 11 amino acid insertion inside of endogenous ingot protein

15:10and then that can reactivate a split luciferase reporter and then the amount of luciferase signal is directly

15:16proportional in theory to how much endogenous syngap is made in

15:22those neurons and so this is all on top of this mouse here uh this this fight this background race

15:28mouse is is crossed onto a homozygous uh um background of a singap one conditional

15:34allele so we can induce uh we can destroy one copy of singap1 in this culture system

15:40by just adding a little bit of aav virus that expresses creatine to induce haploid sufficiency so this system checks all the boxes for

15:47a good disease modeling assay and here is just some data showing that the luciferase reporter does a very good

15:53job at tracking uh uh in a very very easy in straightforward way

15:58uh in a very highly quantitative way the amount of endogenous synthetics made so this is roughly where we uh inserted the

16:04luciferase reporter uh this shows you that the insertion of this reporter doesn’t uh change

16:09endogenous syngap expression but here you see in the in the heterozygous knock in mice uh

16:15this tag also includes not only a luciferase tag but a flag tag and you can see that you only see

16:20a signal in the heterozygous knock in mice that have the tag but importantly it’s the correct length 135 kilodalton

16:26and you can also show that this we can use the luciferase reporter on uh on immunoblot to show that it’s

16:32also present only in these knocking mice this is a quantification of synga protein total syngap protein from this

16:38mice importantly we don’t see a draw a change in endogenous thing f expression so the tag appears benign uh at least in terms of syngap

16:46expression and then we did uh correlations between uh how much protein we add and exactly how

16:52much signal we get both with the singap antibody as well as uh this this end bit signal which is a luciferase

16:57reporter and you see that the the reporter does as good of a job as seeing

17:02antibodies to report the relative amount of syngap protein that’s present so we’re pretty excited about this mouse

17:08and then we had to take it and adapt it to high throughput screening assay plates and so what we do is we take

17:13assay plates that have primary cultured neurons in them and each acid plate has 384 wells

17:20uh and so what you could then do is then use a high throughput a very homogeneous

17:26luciferase acid that takes just you know a couple of steps in 10 minutes and what you end up with is you get uh in this uh

17:33in in these acid plates you get uh 15 to 20 000 units of fluorescence sometimes even more

17:39uh which results in an incredibly high signal to noise for syngap protein expression over background and

17:45gives us metrics that are indeed more than what we need to do high throughput importantly in this assay

17:51format we wanted to know if it can tell us relatively how much syn gap is there with relative uh precision and so what we did is we

17:58plated either heterozygous or homozygous luciferase reporter knocking mice and we

18:03would expect if this worked well you’d get about twice as much signal in the homozygous mice because they have two copies of the tag same gap relative

18:09to one and in fact that’s exactly what we see we see uh you know essentially a doubling in the luciferase signal

18:16indicating that we think we have a pretty good system for for tracking in a very easy uh and

18:21straightforward way the amount of relative syngap expression so we have a good assay and

18:26we can do this in primary neurons but the problem is is that you need a facility and you need to convince this facility to take

18:32your your system and adapt it to high throughput luckily scripps has one of the uh most well-respected

18:38academic high-throughput screening centers they’ve done more than 300 full deck million compound

18:43micro hts screens across more than 100 nih grants and you know over 100

18:50papers there have been 77 identified molecular probes so that’s meaning that new compounds that can modify

18:56a particular protein to to regulate cell biology and importantly scripps has more than a dozen fda approved medicines most

19:02recently to families in azonomon and azonomon is interesting is that actually came from

19:08the libraries one of the libraries in the scripps high-tech screening center that i’m using and we will indeed

19:14be using a lot of the same libraries to look to see if they can regulate syngap expression so the important thing is we have a

19:19facility that has a track record for doing high throughput screening and identifying drug candidates

19:26and so what i had to do is convince the team in the screening center was to

19:31take this mouse-based system we have and to adapt it to to their high

19:36throughput industrial robotics and so we published a paper a few years ago showing that we uh in collaboration uh with tim

19:44spicer and louis scampi who are the directors of the scripps screening center that indeed were able to take uh this this mouse-based primary neuron

19:50system and scale it up to uh assay plates that are uh compatible with uh with high

19:56throughput screening so here here we use genetic knock and mindstate express uh synaptic reporters because

20:02this allowed us to not only test the ability to if we can do it but how well can we track the development

20:07of a metric of a healthy neuronal culture and so synapses are one way to do that and we see that we see very low variance

20:13between wells uh in an assay plate that made us very happy and the synapses are much harder to measure

20:18than than say luciferase which is which is a homogeneous signal from from each well so we were very excited about this so we

20:25are able to plate neurons in such a way that they can be used on high throughput robotics but how can we get enough

20:32throughput in order to screen the you know 100 000 compounds that we think we would need to screen in order to find

20:37robust synthetic regulators and that can be easily overcome by a reasonable size mouse breeding colony where we get just two to four

20:44liters per week uh and so within those uh call you know if you use uh eva 384 or even a 15 36 well plate

20:52we’re talking very easily with just a few hours of work a week you can you can do thousands and thousands of assays uh a week so over the course of

20:59uh you know a few dozen weeks you can get to that hundred or two hundred thousand compounds uh relatively uh in a relatively

21:06painless way so what we did is once we we convinced the facility to help to collaborate with

21:11us to to convert this to high throughput screening uh we did a pilot screen so you want to know you say yes so

21:16yes your assay may work uh by showing you that there’s more or less syngap expression in there when you do heterozygous or homozygous knocking mice

21:23but what how does it perform when you do an actual library so in order to test the you know the it’s more of a dress rehearsal for how

21:29well does my assay work in high throughput screening as we did we use the thousand compound scout library and this library is

21:34interesting is it’s made up of very small molecules that have that was designed by a colleague of mine chris parker

21:40where all of the the constituents of this library are known to bind within binding pockets for

21:45druggable pathways and so some of these compounds many of these compounds have also a

21:51diazerine moiety and diazerines are interesting because they can using uv light you can covalently couple

21:56the compound to the protein it binds to it makes it much easier to figure out the mechanism for how you may be changing the biology

22:03that you’re interested in this case changing sync up one levels and so what we did is we did this very easily just in in one or two weeks

22:10and we did each assay each compound thousand compounds each compound was done in quadruplicate to understand how reproducible the data

22:17are in this format so what did we find so remember there’s a background luciferase this background luciferase tells you how

22:23many neurons are present and that’s important because we want to know uh we want to know if these compounds

22:29are toxic or just generally regulate singap expression so for instance anything that dramatically increases or

22:34decreases syngap expression in this background firefly luciferase you know would be less interesting to us

22:39because it might just be doing something wonky to the cells so in every assay plate we have dmso controls which are in this band

22:45and this this gray shaded region here is what we call the hit index so anything that falls outside of this grey

22:51shaded region is significantly regulating uh uh the reporter in this case firefighter

22:56separates so in dmso control we wouldn’t expect any outside this band and we don’t do that which tells us that we don’t get any false positive which is

23:02great all these dots are the thousand compounds there’s a thousand dots here which represents each unique compound

23:08and you see that indeed you have some increase in some decrease in the background the separated signal which is not not expected

23:14but for every well we have two signals we have the background luciferase but that will also contains

23:19a signal for syngap which is this n-bit signal so what we’re able to do then is to try to compare the results across both

23:26on both data points to find those compounds that regulate the syngap signal but don’t do anything to the firefly signal

23:32this inset here is important as it shows that these were and each data point you see here is four

23:37individual assay wells for the same compound there are no error bars because essentially there’s absolutely no variance so this is

23:43uh say data from plate 1 versus data from plate 2 and you see almost a straight line meaning that we get

23:49essentially the same data we get the same signal across every plate so that tells us that

23:54we really only need to screen in one or two wells when we do the high throughput screen now what did we find in this pilot

24:00screen because it’s possible in the first thousand compounds that we may find something interesting and indeed we found several interesting things so

24:07because of the ability to uh to uh plot the syngap signal for each

24:12data point versus the background signal what we’re looking for are compounds that basically uh up or

24:19down regulate the synthetic signal with having no effect uh on on the luciferase signal

24:24and indeed uh and this would be present in this band here right so if you see a compound way up here

24:29that would be a fifty percent increase in the syngap signal uh with no change uh in the in the luciferase

24:35unfortunately uh it’s always much harder to find up regulators of any biological pathway we found a couple that were

24:41borderline hits but what but because this was a pilot screen and we were really testing the chops of our assay in our system we were we focused

24:48on these what looked like selective down regulators and so down regulators are interesting in the sense that they could be inhibiting

24:54pathways uh they could be regulating pathways that can depress syngap but if you are able to manipulate those

25:00pathways in the opposite direction you may be able to then take that pathway and then trick it into making more syngap and so that’s why we think

25:06it’s important to not only find these compounds and then figure out how they work what the mechanisms are because the whole point of this is to

25:12elucidate uh potential druggable pathways that can be used to change synga singup expression okay so we found seven

25:20or eight potential down regulators and so then when one did we then did is did a follow-up dose response study

25:26so this is the typical drug discovery workflow is you take your your hip from your high

25:31throughput assay you get fresh compound you dissolve it and then you do a concentration response curve and interestingly what we

25:38found is is that in this in this dose range here where we see no effect in the background luciferase we again

25:44repeat our data where we see a dose-dependent increase or i’m sorry a dose dependent increase

25:49in syngp inhibition and we see this of five of the seven compounds which was pretty exciting to

25:54us indicating that our screen is pretty good at pulling out compounds uh that actually move the reporter uh in

26:01a particular direction and it’s reproducible so we’re not going to get a lot of false uh noisy uh data points from our high

26:07throughput screen thank you i’m almost done appreciate it

26:12perfect yeah and so uh we were particularly excited by this is because i mentioned that some compounds were

26:18plus and minus the diazerine uh covalent modification motif and in fact this uh this uh two one nine one

26:25four uh two one nine one five four compound is indeed the compound plus and minus

26:30diazerine in the co in the in the uh library was designed in such a way that diazerines would be would should not

26:36interfere with binding pockets so that told us that you know we pulled out both of these so this is a particularly interesting

26:42compound to us so we’re pretty excited about our assay we were just getting ready to do screening uh at high throughput

26:49for uh all of 2020 but of course covid got in the way of that so we are just now getting back up to speed and so

26:56to give you an idea of how the screen is going to work it’s going to encompass pretty much all of 2021

27:02where we do one uh litter per week per staff member and we have two current staff and so if we do each compound and

27:08duplicate as i mentioned if you do the math for these three four well plates you can come up with after 40 weeks of

27:14iterative screening of 102 000 compounds and so that’s important because 102 thousand compounds

27:20based on prior data from different types of high throughput screens should yield uh several dozen um validated syngap

27:28uh regulators to choose from to maybe most efficacious and then the last thing i’ll the last

27:33slide i’ll leave you with is that then it’s one thing to be able to find these compounds but as i mentioned you have to

27:39have a system uh and an environment to be able to validate them and so

27:44most of what my lab is known for is developing models to understand how synthetic paper insufficiency

27:49affects neural structure and function so we can use that knowledge as ways to validate

27:54uh as a platform for validating the compounds that we pull out so for instance we now have a human ipsc model so if we found a

28:01compound that raises synga protein and mouse neurons so does that also raise synga protein and human neurons does it raise

28:07it in human neurons that come from ipscs from patients for instance and then what we would want to do is take

28:13those compounds and try to reverse uh uh of various uh cellular and network

28:18measures that we know uh from our mouse models uh results from singaporean sufficiency haploid sufficiency such as changes in

28:24synapse function uh memory uh and behavior and so with that i would like to just thank

28:30um for this project in particular uh the co-directors of the of the screening libraries uh center at scripps florida

28:37nih has been funding this now for for three cycles uh uh and as well uh as other funders

28:43and in particular uh i’d like to thank singap research fund for funding a postdoc in the lab uh while they’re not directly uh

28:50involved in this project they are gonna be involved certainly in in studies uh to validate

28:55the compounds that we pull out from the primary screen thank you very much

29:01thanks kevin that was wonderful that is really exciting work um and we’re excited for 2021

29:10to see what comes out of this um there are a couple questions in the q a we have time for i think one or two i think uh

29:18you answered janie’s question during your talk she says i typed that too early so um janie please please update if

29:26part of it wasn’t answered um and there’s a question about whether the compounds that you’re

29:31testing in this library are known to cross the blood-brain barrier

29:37but do you have an idea how many are yeah so we there’s a million compounds to choose from and that’s not an exaggeration there’s

29:44literally you know more than a million compounds and so one of the jobs of the center is to is to match the

29:49sublibraries to the project you want and so there are some you know on the face of it you would say well it’s a

29:54brain disorder you want to only screen compounds that have uh you know blood brain barrier penetrance however

30:00medicinal we also work with medicinal chemists and they tell me that that’s not a big barrier that if you have an

30:05active compound a backbone that that does not cause verbal and bear you can do uh downstream uh

30:11derivatization to to help them cross but i should also mention that’s one of the reasons why you want to screen so many

30:16compounds is you need to have what are called different uh chemical structures or backbones to work with right so some of them

30:22may be really great in in cells to regulating syngap expression but no matter what you do you can’t get it to cross bbb i mean that’s a dead

30:28candidate so you need then you need to go to canada two candidate three candidate four so right well it’s great that you can

30:34screen so many that’s a really nice high-throughput system um and then one last question um in addition to finding drugs that

30:41modulate the disease can you use your system to validate asos or other similar treatments yeah

30:47that’s a good question so you know i’ve given this talk a few times and i always forget so i forgot to mention and also this is relevant to um

30:54to srs so one of the things our srf fellow is doing is to uh we’ve created the same

31:00luciferase screening platform we made a variant uh we made an insertion into

31:06an ips c lounge ipsc line that comes from a patient and we did that allele specifically so we put the reporter tag into uh uh

31:13you know the un the non-mutated syngap allele and it reports syngap protein really nicely in developing human neurons and so

31:20um while this platform pers while my screen per se won’t be good for that we were developing tools

31:26to support the platform that could be generally useful for the community to be able to track syngap protein so so

31:32if somebody was interested in asos uh you know they could have our ipsc patient cell line and they could they

31:37would have a very easy way of tracking uh endogenous to get protein expression

31:43that’s great thank you um perfect i think uh the questions were answered and we

31:48should probably move on to our next

31:58speaker